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1.
FIGURE 6.

FIGURE 6. From: The Endoplasmic Reticulum Stress Response Factor CHOP-10 Protects against Hypoxia-induced Neuronal Death.

Enforced CHOP-10 expression protects neurons against hypoxia. A, Western blotting confirms amplicon vectors encoding full-length (WT) and truncated forms of CHOP lacking the leucine zipper (LZ−) are expressed after transfection in HEK293 cells. B, DIV6 cortical neuronal cultures were transduced with amplicon virus (multiplicity of infection 0.7) expressing full-length CHOP, the LZ− mutant, or luciferase as a control 12 h prior to hypoxic challenge. Levels of nuclear pyknosis were assessed in non-transduced (no virus) or virally transduced cultures exposed control and hypoxic conditions (24 h, 0.5% O2). Results are presented as the average ± S.D. levels of nuclear pyknosis (*, p < 0.05; **, p < 0.01) after subtracting background levels of spontaneous nuclear pyknosis.

Marc W. Halterman, et al. J Biol Chem. 2010 July 9;285(28):21329-21340.
2.
FIGURE 4.

FIGURE 4. From: The Endoplasmic Reticulum Stress Response Factor CHOP-10 Protects against Hypoxia-induced Neuronal Death.

Effects of CHOP deletion on neuron survival. A, detection of CHOP-10 protein by Western blotting in wild-type but not knock-out (KO) cultures following Tg exposure (1 μg/ml, 24 h; DMSO vehicle control shown). B, pyknosis responses of wild-type (WT) and KO cultures exposed to DMSO (−) or Tg (1 μg/ml). C, survival analysis in WT and KO cultures exposed to DMSO (open bar) or two Tg doses (10–100 ng/ml; n = 6 per condition). D, profiles of nuclear pyknosis in WT and KO cultures induced by hypoxic challenge (0.3% versus 0.1% O2, 24 h). Results represent the average ± S.D. Significance testing as performed by analysis of variance with Newman-Keuls multiple comparison test for post-hoc analyses is shown (*, p < 0.05; **, p < 0.01; and ***, p < 0.001). E, Western analysis for levels of cleaved caspase-3 (cCasp3) in WT and KO cultures.

Marc W. Halterman, et al. J Biol Chem. 2010 July 9;285(28):21329-21340.
3.
FIGURE 7.

FIGURE 7. From: The Endoplasmic Reticulum Stress Response Factor CHOP-10 Protects against Hypoxia-induced Neuronal Death.

CHOP is required for BDNF-mediated protection. A, BDNF (50 ng/ml) pretreatment (12 h, n = 3) protects cortical neurons against hypoxic injury (0.5% O2, 24 h). B, Western analysis of control and BDNF-treated (50 ng/ml) wild-type primary cortical cultures before and after hypoxic challenge (0.3% O2, 24 h). C, wild-type and CHOP knock-out cultures were treated with BDNF as above and analyzed by Hoechst analysis after incubation at normoxic (N and N+B) or hypoxic (H and H+B) conditions. Survival data were derived from an average of 250 counts per coverslip (n = 3; average ± S.D.). D, analysis of lysates from C for levels of cleaved PARP and actin. Control samples exposed to either DMSO (ctrl) or thapsigargin (Tg, 100 ng/ml) are shown. Significance testing was performed by analysis of variance with Newman-Keuls multiple comparison test for post-hoc analyses (*, p < 0.05; ***, p < 0.001).

Marc W. Halterman, et al. J Biol Chem. 2010 July 9;285(28):21329-21340.
4.
FIGURE 2.

FIGURE 2. From: The Endoplasmic Reticulum Stress Response Factor CHOP-10 Protects against Hypoxia-induced Neuronal Death.

Validation of gene expression in hypoxic cortical neuron cultures. A, quantitative PCR validation of hypoxia-induced microarray targets. RNA prepared from normoxic controls (0 h) or samples exposed to between 3 and 18 h of hypoxia (0.5% O2) was subjected qPCR for the targets vegf, glut-1, hkII, egr-1, atf5, and chop-10. The -fold change in mRNA expression (average ± S.D.) relative to normoxic controls using the ΔΔCT method is shown with significance measured by Student's t testing (*, p < 0.05; **, p < 0.01; and ***, p < 0.005; hypoxic versus paired normoxic controls). B, hypoxia activates ER stress and apoptotic targets in cortical cultures exposed to continuous hypoxia (0.5% O2). Samples were analyzed at the indicated times by Western blotting for the following targets: phosphorylated and total eIF2α (p-eIF2α), ATF4, CHOP-10, cleaved PARP (cPARP), cleaved caspase-3 (cCasp3), and actin.

Marc W. Halterman, et al. J Biol Chem. 2010 July 9;285(28):21329-21340.
5.
FIGURE 3.

FIGURE 3. From: The Endoplasmic Reticulum Stress Response Factor CHOP-10 Protects against Hypoxia-induced Neuronal Death.

Immunocytochemical analysis of ATF4 and CHOP expression. A, hypoxia-induced ATF4 expression co-localizes with nuclear condensation in neuronal cultures (0.5% O2, 24 h, left panels). Expression of the ATF4 target Tribbles 3 (TRB3) co-localizes with nuclear ATF4 in exposed cultures (top right). Time-dependent increase in ATF4 and TRB3 expression in hypoxic cultures is shown. Data are expressed as the fraction of cells expressing ATF4 or TRB3 (12–24 h, 0.5% O2) relative to untreated controls from an average of 150 cells per coverslip (n = 3). B, broad pattern of CHOP expression in thapsigargin (Tg)-treated cortical cultures. Normoxic cultures were treated with 1 μg/ml Tg for 24 h and analyzed by immunocytochemical analysis using the B-3 monoclonal CHOP antibody. C, a distinct pool of CHOP-10 was expressed widely in cultured embryonic neurons. Double immunocytochemical analysis was performed using the CHOP-reactive polyclonal antibody F-168 and the monoclonal anti-neuronal marker NeuN. Results using CHOP knock-out cultures demonstrate only faint perinuclear staining with the F-168 antisera (scale bar = 20 μm).

Marc W. Halterman, et al. J Biol Chem. 2010 July 9;285(28):21329-21340.
6.
FIGURE 1.

FIGURE 1. From: The Endoplasmic Reticulum Stress Response Factor CHOP-10 Protects against Hypoxia-induced Neuronal Death.

Temporal analysis of adaptive and apoptotic gene expression in hypoxic neuronal cultures. A, schematic of the delayed delivery exposure paradigm. To determine the temporal window of pathological transcription and translation required to stimulate nuclear pyknosis in vitro, dissociated cortical cultures maintained under either normoxic (open bars) or hypoxic conditions (filled bars, 0.5% O2), were treated with either vehicle (DMSO), the transcriptional inhibitor actinomycin D (Act D), or the translation inhibitor cycloheximide (CHX) at various points after the onset of hypoxia. B and C, delayed addition of actinomycin D (1 μg/ml in DMSO), or CHX (200 nm) enhances neuronal cell survival in vitro. Cultures were assessed for levels of nuclear pyknosis after 24-h exposure; data are expressed as the absolute fraction of pyknotic nuclei with significance measured by Student's t testing (average ± S.D.; *, p < 0.05; **, p < 0.01; and ***, p < 0.005).

Marc W. Halterman, et al. J Biol Chem. 2010 July 9;285(28):21329-21340.
7.
FIGURE 5.

FIGURE 5. From: The Endoplasmic Reticulum Stress Response Factor CHOP-10 Protects against Hypoxia-induced Neuronal Death.

Effects of shRNA-mediated CHOP-10 knockdown on neuron survival. A, pPRIME-CHOP lentiviral transfer vectors were transfected into N2A cells along with the expression vector eGFP-CHOP-V1 and incubated for 72 h before harvesting lysates. Levels of the expressed fusion protein and endogenous CHOP levels were measured by Western blotting and compared with the luciferase targeting control vector pPRIME-FF3 for efficiency of gene silencing. B, fluorescence analysis of GFP expression and nuclear pyknosis in cultures transduced with GFP-expressing pPRIME vectors. C, primary cultures (DIV4) were transduced with either pPRIME-FF3 or pPRIME-CH-B1 lentivirus (multiplicity of infection 0.25), exposed 2 days later to hypoxia (24 h, 0.3% O2, 24 h), and analyzed for the number of surviving GFP-positive cells. D, primary cultures (DIV4) were transduced with pPRIME-FF3 or pPRIME-CH-B1 lentivirus (multiplicity of infection 0.75) and exposed to hypoxia (DIV6, 24 h, 0.3% O2) prior to nuclear pyknosis analysis (DIV9). Results are presented as the average ± S.D. levels of nuclear pyknosis. Significance testing was performed by analysis of variance with Newman-Keuls multiple comparison test for post-hoc analyses (*, p < 0.05).

Marc W. Halterman, et al. J Biol Chem. 2010 July 9;285(28):21329-21340.

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